The loss of blood or plasma caused by surgery or injuries can be compensated by infusions of whole blood, or by the infusion of blood substitutes or plasma extenders which are capable of oxygen transfer.
Due to the well known problems related to the cost, availability and safety of whole blood, attempts have been made to provide blood substitutes capable of transferring oxygen. There are currently two basic approaches towards achieving this end.
First, fluorocarbons may be used as gas transporting substitutes for blood (Med. Welt 32:1338 (1981) and Med. Lab. Sci. 39:45 (1982)). A disadvantage of the fluorocarbon emulsions is that they are not completely eliminated from the body, nor are they degraded. Instead they accumulate in organs such as the liver or spleen and remain for at least several years. Widespread use of fluorocarbons is further hindered by the fact that an oxygen-rich atmosphere is required for respiration. Fluorocarbons are also unstable in storage.
The second approach is to use hemoglobin-containing solutions with an oxygen-transferring function as blood substitutes or plasma extenders. Ordinary solutions of stroma-free hemoglobin are not suitable as blood substitutes because of their rapid physiological turnover. Hemoglobin which is not bound by erythrocytes decomposes relatively quickly into its sub-units; these sub-units have a molecular weight of less than 64,500 daltons, and are promptly eliminated by the kidneys.
There have been attempts at improving the efficacy of this second approach by preventing the dissociation of hemoglobin into its sub-units, thereby increasing the intravasal half life. For instance, hemoglobin has been cross-linked intramolecularly or intermolecularly with suitable bifunctional cross-linking agents so as to obtain a molecule size which will ensure a longer residence in the circulation. In this regard, German Offenlegenschrift DE-OS No. 24 17 619 describes plasma protein substitutes which contain hemoglobin where the sub-units are cross-linked to polymers of mean molecular weights between 68,000 and 600,000 with the aid of dialkyl dicarboxylic acid imidates. Alternatively crosslinkage may occur intermolecularly between different hemoglobin tetramers or simultaneously intra- and/or intermolecularly
German Offenlegenschrift DE-OS No. 26 07 706 also discloses water-soluble hemoglobins, the sub-units of which are cross-linked to polymers with molecular weights of 64,000 to 1,000,000 by means of suitable cross-linking agents, including triazines, multi-substituted polyfunctional benzene derivatives, linear or cyclic polyfunctional poly-alkylene derivatives, dicarboxylic acid derivatives, or dialdehydes. Intermolecular cross-linking of hemoglobin tetramers is also described. The subject of Published European Patent Application EP-A No. 81 301 535.1 is a method for producing artificial red blood cells which contain aqueous, stroma-free hemoglobin solutions in membranes of polymerized hemoglobin. The crosslinking agents of German Offenleigen-schrift DE-OS No. 26 07 706 are used to polymerize hemoglobin and thus to prevent its rapid degradation in the body.
Another way to increase the intravasal half life of stroma-free hemoglobin in the body is to bind hemoglobin covalently to physiologically safe high-polymer materials. Physiologically compatible polymers include hydroxy-ethyl starch (German Offenlegenschrift DE-OS No. 26 16 086) and other polysaccharides which are covalently bound by way of bridges (German Offenlegenschrift DE-OS No. 30 29 307); insulin (published European patent application EP-A No. 81 302 858.6), dextran (Thamm, et al., Proc. Nat. Acad. Sci. USA, 73:2128 (1976); Humphries et al., Proc. B.P.S., 191 (1981); Baldwin et al., Tetrahedron 37:1723 (1981); and polyethylene glycols of different molecular weights (Ajisaka et al., Bioch. Biophys. Res. Comm. 97:1076 (1980); U.S. Pat. No. 4,301,144).
All of the known hemoglobin-containing molecules with high molecular weight share the problem that the covalent bond between the hemoglobin and the crosslinking agent or polymer changes the quaternary structure of that protein component of the hemoglobin which is responsible for oxygen transport, and thus adversely affects the natural properties of the hemoglobin. Although such deoxygenated hemoglobin can bind oxygen, the oxygenated hemoglobin can no longer adequately release the bound oxygen in the peripheral tissue. This increase in oxygen affinity is demonstrated by a shift of the central portion of the oxygen dissociation curve to the left, so that the oxygen dissociation curve for natural hemoglobin represents a lower percentage of oxygen saturation at a given oxygen partial pressure than the corresponding curves for covalently bound hemoglobin compounds as described in the art, which more nearly resemble the hyperbolic curve for myoglobin than the curve for natural hemoglobin.
As the quaternary structure of natural hemoglobin is disturbed by covalent bonds between the protein component and the crosslinking agents or polymer molecules, these cross-linked or polymer-bound hemoglobins show a reduced subunit cooperativity with respect to the binding ability for oxygen. The effect can be measured quantitatively in terms of the reduction in the Hill coefficient, which decreases from 2.8 to lower values. Thus, the curve for cross-linked or polymer-bound hemoglobin derivatives bears greater resemblance to the hyperbolic form of the curve for myoglobin than to the sigmoidal curve form typical of functionally intact hemoglobin.
The relationship between an increase in molecular size brought about by cross-linkage or attachment of the hemoglobin to polymers and an undesired increase in oxygen affinity makes this approach disadvantageous for use as a blood substitute. This disadvantage cannot be eliminated by attaching allosteric effectors to the phosphate bond site of cross-linked hemoglobin in an effort thus to improve the oxygen-yielding ability of the cross-linked or polymer-bound hemoglobins. German Offenlegenschrift DE-OS No. 27 14 252 describes methods for cross-linking hemoglobin with dialdehydes where the hemoglobin contains pyridoxal-5-phosphate instead of the allosteric modulator 2,3 - diphospeoglyceate naturally bound to the phosphate bond site.
The method of German Offenlegenschrift DE-OS No. 31 44 705 follows the same approach, using inositol hexaphosphate as allosteric modulator. While the introduction of the modulator improves the oxygen-yielding ability, the allosterically modified hemoglobin preparations suffer from the disadvantages that result from intramolecular cross-linkage of the hemoglobin sub-units, or intermolecular cross-linkage of hemoglobin tetramers. None of the preparations known in the art are suitable for broad therapeutic use.
Thus, there remains a need for a hemoglobin-containing blood substitute which is suitable as an oxygen and carbon dioxide carrier, is capable of oxygen uptake and release under natural conditions, and does not have the foregoing disadvantages.